Low-autofluorescence and low-reflectance optical components for microscopes, and microscopes utilizing same

ABSTRACT

An optical component for a microscope may include a low-autofluorescence substrate, or a substrate and a high-performance anti-reflective layer coating the substrate. An optical component may include a low-autofluorescence substrate and high-performance anti-reflective layer coating the low-autofluorescence substrate. The high-performance anti-reflective layer may be a low-autofluorescence high-performance anti-reflective layer. A microscope may include one or more such optical components.

RELATED APPLICATION

This application claims priority to U.S. provisional application No.62/145,347 filed on Apr. 9, 2015, the content of which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

The present invention relates generally to confocal microscopy andparticularly to spinning disks utilized in confocal microscopy.

BACKGROUND

Microscopes are employed in a wide variety of fields to view varioustypes of biological and non-biological samples. In some applications,for example in certain applications of life sciences, confocalmicroscopes are preferred over more traditional wide-field microscopes.In a confocal microscope, light is passed through a small aperture(traditionally a pinhole) positioned at an optically conjugate plane.The point illumination substantially eliminates out-of-focus light(background signal) and thereby increases the optical resolution andcontrast of the image acquired. However, these advantages are realizedat the expense of decreased light intensity caused by the confocalaperture, such that longer exposure times are often required incomparison to wide-field microscopes.

As only a single small point on the sample is illuminated at any time,the confocal microscope often provides a scanning function. One type ofscanning technique entails the use of a spinning disk, also known as ascanning disk or Nipkow disk. A typical spinning disk includes multipleapertures arranged along one or more spiral paths. Scanning isimplemented by spinning the disk at a high angular velocity, for examplethousands of revolutions per minute (RPM).

Two problems associated with microscopes, including confocal microscopessuch as spinning disk microscopes, are reflectance of stray light fromoptical components and autofluorescence exhibited by optical components(i.e., generation of secondary stray light in an optical component).Reflected stray light includes light that is not part of the image ofthe sample and, in the case of fluorescence microscopy, light that isnot part of the fluorescent response of the sample. Autofluorescence inoptical components is generated by color centers in such opticalcomponents. The color centers are due to the presence of rare earthelements and other impurities in the material (e.g., optical glass orpolymer) of the optical components. Optical components that may exhibitunacceptably high reflectance and autofluorescence include the spinningdisk as well as lenses and windows positioned on the optical axis of themicroscope.

Conventionally, these problems have been addressed by coating suchoptical components with a conventional material specified as being alow-reflectance material or an anti-reflective material. Additionally,the microscope may include an emission filter effective for blocking alarge portion of unwanted light. Nevertheless, even when such measuresare taken an unacceptable amount of stray light andautofluorescence-based light may reach the imaging device of themicroscope. This “ghost” light has the appearance of background inconfocal images, thus lowering signal-to-background ratio for a givenexposure time or increasing the exposure time required to attain adesired signal-to-background ratio. Therefore, it would be desirable toreduce the amount of stray light reflected from optical components andlight emitted from optical components due to autofluorescence.

SUMMARY

To address the foregoing problems, in whole or in part, and/or otherproblems that may have been observed by persons skilled in the art, thepresent disclosure provides methods, processes, systems, apparatus,instruments, and/or devices, as described by way of example inimplementations set forth below.

According to one embodiment, an optical component for a microscopeincludes a low-autofluorescence substrate.

According to another embodiment, an optical component for a microscopeincludes a substrate and a high-performance anti-reflective layercoating the substrate.

According to another embodiment, an optical component for a microscopeincludes a low-autofluorescence substrate and a high-performanceanti-reflective layer coating the low-autofluorescence substrate.

According to another embodiment, a microscope includes: a light sourceconfigured for producing excitation light; a light detector configuredfor acquiring an image of the sample; and optics defining an excitationlight path from the light source to the sample, and an emission lightpath from the sample to the light detector, wherein the optics comprisean optical component, and the optical component comprises alow-autofluorescence substrate, or a substrate and a high-performanceanti-reflective coating the substrate, or both of the foregoing.

According to another embodiment, a method for acquiring microscopicimages of a sample includes: operating a microscope according to any ofthe embodiments disclosed herein to irradiate a sample with excitationlight and collect emission light emitted by the sample, wherein theexcitation light and/or the emission light passes through the opticalcomponent comprising the low-autofluorescence substrate.

Other devices, apparatus, systems, methods, features and advantages ofthe invention will be or will become apparent to one with skill in theart upon examination of the following figures and detailed description.It is intended that all such additional systems, methods, features andadvantages be included within this description, be within the scope ofthe invention, and be protected by the accompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be better understood by referring to the followingfigures. The components in the figures are not necessarily to scale,emphasis instead being placed upon illustrating the principles of theinvention. In the figures, like reference numerals designatecorresponding parts throughout the different views.

FIG. 1 is a schematic view of an example of a confocal microscopeaccording to some embodiments.

FIG. 2 is a plan view of an example of a spinning disk according to someembodiments.

FIG. 3A is a schematic plan view of a portion of an example of anaperture section of a spinning disk according to some embodiments.

FIG. 3B is a schematic plan view of a portion of an example of anaperture section of a spinning disk according to other embodiments.

FIG. 4 is a schematic cross-sectional view of a spinning disk accordingto some embodiments.

FIG. 5 is a side view of an example of a doublet according to someembodiments.

DETAILED DESCRIPTION

FIG. 1 is a schematic view of an example of a confocal microscope 100according to some embodiments. Generally, the structure and operation ofvarious types of microscopes are understood by persons skilled in theart, and thus certain components and features of the microscope 100 aredescribed only briefly to facilitate an understanding of the subjectmatter taught herein. The microscope 100 may generally include a lightsource 104 configured for generating excitation light 106, a samplestage 108 for supporting a sample under analysis, a light detector (orimage sensor) 112 configured for generating excitation light 114,various optics for defining (i.e., establishing or providing) anexcitation (or illumination) light path from the light source 104 to thesample, various optics for defining an emission (or detection) lightpath from the sample to the light detector 112, and a confocal spinningdisk 116 (also termed a scanning disk) positionable in an intermediateimage plane and simultaneously in the excitation light path and theemission light path. In some embodiments, the confocal microscope 100may be configured for detecting light reflected (scattered) from thesample at the same wavelength as the light utilized to illuminate thesample. In other embodiments, the confocal microscope 100 may beconfigured for exciting the sample at a specific excitation wavelengthand detecting fluorescent light emitted from the sample at a longerwavelength in response to the excitation. In these latter embodiments, afluorescent agent or fluorophore (e.g., fluoroscein) is added to thesample as appreciated by persons skilled in the art. For convenience,unless specified otherwise or the context dictates otherwise, the term“excitation” encompasses “illumination” of a sample for the purpose ofcollecting reflected or scattered light, as well as excitation of asample for the purpose of collecting fluorescent emission light emittedby the sample.

The sample stage 108 is schematically depicted as a sample plane atwhich the sample is positioned by the sample stage 108. The sample stage108 may generally be any platform for supporting a sample, or a sampleand a substrate supporting the sample (e.g., a glass slide), in a fixedposition thereon. In some embodiments, the sample stage 108 may bemovable by manual or motorized actuation. That is, the position of thesample stage 108 may be adjustable by the user along the x-axis, y-axis,and/or z-axis. In the present context, the z-axis is taken to be theoptical axis that is orthogonal to the sample plane, and the x-axis andy-axis are taken to lie in the sample plane. The sample may generally beany object for which imaging is desired and which is mountable to thesample stage 108. The sample may be biological (e.g., spores, fungi,molds, bacteria, viruses, biological cells or intracellular components,biologically derived particles such as skin cells, detritus, etc.) ornon-biological (e.g., chemical compound, particulate matter, etc.).

The light source 104 may be any light source suitable for confocalmicroscopy. For example, the light source 104 may be a solid-state lightsource such as a light emitting diode (LED) or a solid-state laser, oralternatively may be a semiconductor-based laser (laser diode or LD). Insome embodiments, the optical output of the light source 104 may be thetip of an optical fiber that is imaged onto (conjugate to) theintermediate image plane where the spinning disk 116 is positioned andthe sample plane where the sample is positioned. In some embodiments,the light source 104 may include a plurality of light sources (e.g.,multiple LEDs) that generate light at different wavelengths. Such lightsources may be mounted to wavelength selector, such as a motorized wheel(not shown) that enables automatic (computerized) selection of the lightsource and thus the wavelength of the excitation light to be utilized ina given application.

The light detector 112 may be any imaging device suitable for confocalmicroscopy such as, for example, the type of imaging device that formsthe basis of cameras. In typical embodiments, the light detector 112 isa multi-pixel (or pixelated) imaging device such as, for example, acharge-coupled device (CCD) or an active-pixel sensor (APS) based oncomplementary metal-oxide-semiconductor (CMOS) technology. In someembodiments, the confocal microscope 100 may include an eyepiece (notseparately shown) to enable the user to view the sample, in which caseappropriate optical components (e.g., beam splitter) are provided tosplit the emission light path so that the emission light is directed toboth the light detector 112 and the eyepiece. Thus, the light detector112 in FIG. 1 may be considered as schematically representing an imagingdevice, or both an imaging device and an eyepiece. Alternatively, thelight detector 112 may be a broadband light source that operates inconjunction with an excitation filter (not shown) that only allows thedesired excitation wavelength of light to pass.

The intermediate optics (optical components) involved in defining theexcitation light path and/or emission light path vary widely from oneembodiment to another. Depending on the particular optical component,defining a light path may include modifying the light beam in somefashion, such as focusing, inverting, or resizing the light beam, etc.Examples of optics may include, but are not limited to, the opticsillustrated in FIG. 1, which are briefly described as follows. In thefollowing description and as used herein generally, the term “lens” mayrefer to either a single lens or a lens group (a series of lenses),depending on the embodiment and the function of the lens(es), asappreciated by persons skilled in the art.

The excitation light generated by the light source 104 is collimated bya collimating lens 120 and is then incident on a long-pass beam splitter124 such as a dichroic beam splitter or mirror. The beam splitter 124 isconfigured to reflect light at the wavelengths contemplated for theexcitation light and to transmit light at the wavelengths contemplatedfor the emission light. In some embodiments, the beam splitter 124 mayinclude a plurality of beam splitters having different opticalreflection/transmission characteristics. Such beam splitters may bemounted to wavelength selector, such as a motorized wheel (not shown)that enables automatic (computerized) selection of the beam splitterwith the desired reflection/transmission spectrum. The excitation lightis reflected by the beam splitter 124 at an angle (e.g., ninety degrees)and is directed through a first relay lens 128 that inverts the image ofthe excitation light. The excitation light then passes through a window132 to the front side of the spinning disk 116. The window 132 may beprovided to prevent dusty air from coming into contact with the spinningdisk 116. Small particles can adhere to the spinning disk 116 with aforce great enough to resist centrifugal forces. Moreover, smallparticles can autofluoresce. Thus, the window 132 is useful forpreventing small particles from contributing the image captured by thelight detector 112 as artifacts or background. The portion of theexcitation light passing through apertures of the spinning disk 116 istransmitted through a field lens 136, a tube lens 140, and an objectivelens 144 to the sample on the sample stage 108. The objective lens 144may be any lens or system of lenses configured for transmitting andfocusing the excitation light onto the sample, and collecting theemission light emitted from the sample and focusing the emission lightonto the light detector 112.

In response to the illumination (or excitation in fluorescenceapplications), the sample emits emission light, i.e.,scattered/reflected light, or fluorescent light in fluorescenceapplications. The emission light is transmitted through the objectivelens 144, the tube lens 140, and the field lens 136, and is incident onthe back side of the spinning disk 116. The portion of the excitationlight passing through the apertures of the spinning disk 116 istransmitted through the window 132 and the first relay lens 128. Theemission light is then transmitted through the beam splitter 124 withoutbeing reflected, and through a second relay lens 148. At least influorescence applications, the emission light then passes through anemission filter 152 to block unwanted wavelengths and is focused on theimage plane of the light detector 112. In some embodiments, the emissionfilter 152 may include a plurality of emission filters having differenttransmission characteristics. Such emission filters may be mounted towavelength selector, such as a motorized wheel (not shown) that enablesautomatic (computerized) selection of the filter with the desiredtransmission spectrum.

The spinning disk 116 generally includes a planar first side 156 and anopposing planar second side 160. The spinning disk 116 is mounted to ashaft or spindle 164 coaxial with the central axis of the spinning disk116. Rotation of the shaft 164 is powered by a suitable motor (notshown). Examples of the spinning disk 116 are described further below.In some embodiments, the spinning disk 116 is selectively movable intoand out from the light paths to enable selection between confocal andwide-field operations.

It will be understood that FIG. 1 is a high-level schematic depiction ofan example of the confocal microscope 100 consistent with the presentdisclosure. Other optics, electronics, and mechanical components andstructures not specifically shown in FIG. 1 may be included as neededfor practical implementations.

It will also be understood that the confocal microscope 100 illustratedin FIG. 1 may also include a computing device (not shown) communicatingwith the light detector 112. The computing device may receive imagescaptured by the light detector 112, and digitize and record the images.The computing device may also process captured images as needed fordisplaying the images on a display device such as a computer screen. Thecomputing device may also be configured for processing the images so asto enhance or control the display of the images in a desired manner.Generally for these purposes, the computing device may include hardware(microprocessor, memory, etc.) and software components as appreciated bypersons skilled in the art. For example, the computing device mayinclude a processor such as a main electronic processor providingoverall control, and one or more electronic processors configured fordedicated control operations or specific signal processing tasks (e.g.,a graphics processing unit, or GPU). The computing device may alsoinclude one or more memories (volatile and/or non-volatile) for storingdata and/or software. The computing device may also include one or moredevice drivers for controlling one or more types of user interfacedevices and providing an interface between the user interface devicesand components of the computing device. In addition to a display device,such user interface devices may include other user output devices (e.g.,printer, visual indicators or alerts, audible indicators or alerts, andthe like) and also user input devices (e.g., keyboard, keypad, touchscreen, mouse controller, joystick, trackball, and the like). Thecomputing device may also include one or more types of computer programsor software contained in memory and/or on one or more types ofcomputer-readable media. Computer programs or software may containinstructions (e.g., logic instructions) for performing all or part ofany of the methods disclosed herein. Computer programs or software mayinclude application software and system software. System software mayinclude an operating system (e.g., a Microsoft Windows® operatingsystem) for controlling and managing various functions of the computingdevice, including interaction between hardware and application software.In particular, the operating system may provide a graphical userinterface (GUI) displayable via a user output device, and with which auser may interact with the use of a user input device. The computingdevice may also include one or more data acquisition/signal conditioningcomponents (as may be embodied in hardware, firmware and/or software)for receiving and processing the imaging data captured by the lightdetector 112, including formatting data for presentation in graphicalform by the GUI, generating 3D images, etc.

FIG. 2 is a plan view of an example of a spinning disk 216 according tosome embodiments. The plan view may be of either the first side 156 orthe second side 160 (FIG. 1) of the spinning disk 216. The spinning disk216 generally includes a central section 264 and an outer section 268coaxially surrounding the central section 264. The central section 264may be configured (e.g., as a hub) for attaching the spinning disk 216to a shaft (e.g., shaft 164 in FIG. 1) to enable motorized rotation ofthe spinning disk 216. The shaft may be attached to one side of thecentral section 264 and/or may pass through a central bore 270 of thespinning disk 216. At least a portion of the outer section 268 is anannular aperture section 272 that also coaxially surrounds the centralsection 264. Relative to the central axis of the spinning disk 216, theaperture section 272 generally has an inner radius and an outer radius,and spans a radial distance from the inner radius to the outer radius.The aperture section 272 may be, or be formed by, a patterned mask asdescribed further below. The aperture section 272 includes a pluralityof apertures 276 arranged along a plurality of spiral paths on theplanar face (first side 156 or second side 160) of the spinning disk216. In the illustrated example the spinning disk 216 includesthirty-six spiral paths but in other examples may include less or morethan thirty-six spiral paths. The spiral paths may be arranged adjacentto each other in a multi-start pattern, such that the spiral paths eachstart at a first radius of the spinning disk 216 (e.g., the inner radiusof the aperture section 272) and end at a second radius of the spinningdisk 216 greater than the first radius (e.g., the outer radius of theaperture section 272). In some embodiments, the spiral paths may follow(or substantially follow) Archimedean spirals that may be expressed byr=a+b(θ), where the radius r and angle θ are polar coordinates and theparameters a and b are real numbers. In other embodiments, the spiralpaths may be non-Archimedean.

In some embodiments, each aperture 276 is a continuous spiral slitextending along a corresponding spiral path from the start to the end ofthat spiral path. In other embodiments, each aperture 276 is a spiralsegmented spiral slit extending along a corresponding spiral path fromthe start to the end of that spiral path. In other words, in suchembodiment each aperture 276 is a plurality of curved slits of equalsize (length and width) positioned at equal distances from each otheralong one of the spiral paths from the start to the end of that spiralpath. In such embodiment, the apertures 276 may be multiple series ofslits occupying respective spiral paths. In still other embodiments,each aperture 276 is a plurality of pinholes of equal size (diameter)positioned at equal distances from each other along one of the spiralpaths from the start to the end of that spiral path. That is, theapertures 276 may be multiple series of pinholes occupying respectivespiral paths.

FIG. 3A is a schematic plan view of a portion of an example of anaperture section 372 of a spinning disk according to some embodiments.In this embodiment, the apertures 376 of the spinning disk arecontinuous spiral slits. Adjacent apertures 376 are spaced from eachother by a separation distance (or “step” or “pitch”) indicated as 380in FIG. 3A. In some embodiments, the separation distance 380 is on theorder of millimeters (e.g., 14 mm). In some embodiments, the separationdistance 380 is constant over the entire extent of the spiral pathstaken by the apertures 376, while in other embodiments the separationdistance 380 may vary. In some embodiments, the width of each spiralaperture 376 (the distance between opposing spiral edges of the aperture376, i.e., the dimension transverse to the spiral length of the aperture376) is on the order of micrometers (μm). In one example, the width isin a range from 10 μm to 100 μm. In another example, the width is in arange from 20 μm to 70 μm. In another example, the width is 50 μm. Insome embodiments, the width is constant over the entire length of theaperture 376, while in other embodiments the width may vary.

FIG. 3B is a schematic plan view of a portion of an example of anaperture section 382 of a spinning disk according to other embodiments.In this embodiment, apertures 386 of the spinning disk are pinholesspaced from each other along the multiple spiral paths. In someembodiments, the diameter of the pinholes is on the order ofmicrometers. For example, the diameter of the pinholes may be on thesame scale as the widths of slit apertures 376 (FIG. 3A) given above.

Generally, there is considered to be a trade-off in desirable opticalproperties when selecting between slit apertures and the moretraditional pinhole apertures for spinning disks. Slit apertures mayprovide relatively brighter illumination of the sample and a moreintense emission signal for the light detector 112 (FIG. 1), whereaspinhole apertures may provide relatively better axial (z-axis)resolution. For a spinning disk having a typical pattern oftypically-sized pinholes, the insertion loss of the spinning disk (i.e.,the loss in intensity of transmitted light resulting from the presenceof the spinning disk in the light paths) has been calculated to be ashigh as 94%. Hence for some applications, including fluorescenceapplications, slit apertures may be preferred. In particular, it is seenthat continuous slit apertures provide improved open-area ratio (OAR)and reduced insertion loss. This in turn reduces the exposure timerequired to attain a desired optical signal, thereby reducing imageacquisition time and increasing system throughput.

FIG. 4 is a schematic cross-sectional view of a spinning disk 416according to some embodiments. The spinning disk 416 may include aplanar first side 456, an opposing planar second side 460, and a centralbore 470. The thickness of the spinning disk 416 is defined between thefirst side 456 and the second side 460. The spinning disk 416 mayfurther include a disk-shaped substrate 490 largely dictating theoverall dimensions of the spinning disk 416, and a patterned mask 492.The substrate 490 includes a planar first surface and an opposing planarsecond surface corresponding to the first side 456 and the second side460, respectively. The thickness of the substrate 490 extends from thefirst surface to the second surface along a direction orthogonal to thefirst surface and second surface. In some embodiments, the thickness ofthe substrate 490 is in a range from 0.5 mm to 2 mm. In someembodiments, the substrate 490 may include a central section 464 that isdistinct from the substrate 490 and may be composed of a differentmaterial. The patterned mask 492 includes a spiral pattern orarrangement of apertures 476 as described above. The patterned mask 492disposed on either the first surface or the second surface of thesubstrate 490.

Generally, it is desirable to minimize reflectance of light incident onthe surface of the patterned mask 492 so as to minimize the amount of“ghost” light that contributes to background noise in the confocalimages captured by the light detector 112 (FIG. 1). In some embodiments,the patterned mask 492 may be composed of a suitable optically black(low-reflective) material. Examples of black materials include, but arenot limited to, chrome-based materials, i.e., materials based onchromium oxide (Cr₂O₃) or based on a mixture of chromium (Cr) and Cr₂O₃such as black chrome and blue chrome. Other inorganic compounds oralloys that are suitably black may also be utilized.

In some embodiments, the patterned mask 492 is a thin layer (or film,coating, etc.) having a thickness on the order of micrometers. Forexample, patterned mask 492 may have a thickness in a range from 0.1 μmto 0.3 μm. The patterned mask 492 may be fabricated by any techniquesuitable for its composition and thickness. For example, the patternedmask 492 may be prefabricated and then laminated on the substrate 490.As other examples, the patterned mask 492 may be formed on the substrate490 by a microfabrication process such as, for example, electroplating,vacuum deposition (chemical vapor deposition or CVD, physical vapordeposition or PVD, etc.), evaporation, or by a wet coating technique(e.g., spray coating, dip coating, spin-on coating, etc.). In someembodiments, the patterned mask 492 may be formed by first depositing acontinuous layer and thereafter patterning the layer to form theapertures 476 by any suitable technique such as photolithography (e.g.,masking and etching).

Generally, the substrate 490 may be composed of any opticallytransparent material suitable for use in a confocal microscope. Examplesof materials for the substrate 490 include, but are not limited to,various glasses and quartz (including fused quartz), as well as certainoptically transparent polymers. One non-limiting example of glass isborosilicate glass, such as BOROFLOAT® glasses available from SCHOTTNorth America, Inc., Louisville, Ky., USA.

According to certain embodiments disclosed herein, the substrate 490 isa low-autofluorescence substrate. In such embodiments, the substrate 490is composed of a low-autofluorescence material. Examples oflow-autofluorescence materials include, but are not limited to, glassesavailable from SCHOTT North America, Inc., Louisville, Ky., USA, havingthe product designations N-SF57HT, N-FK51A, N-KZFS8, N-BK7, and N-KZFS5.Generally, the low-autofluorescence material should be a high-puritygrade optical material so as to minimize or eliminate color centers inthe material that result in autofluorescence. In some embodiments, amaterial disclosed herein as being low-autofluorescence material has aconcentration of impurities of transition metals and rare earth elementsof 20 ppm or less.

In some embodiments, to further lower the reflectivity of the spinningdisk 416, the spinning disk 416 may include an anti-reflective (AR)layer or coating 494 that conformally coats (covers) the first side 456and the second side 460. Generally, the anti-reflective layer 494 may beany material or multilayer stack of materials exhibiting effectiveanti-reflective characteristics for the spectrum of wavelengthscontemplated by the present disclosure (e.g., from 350 nm to 700 nm, orabout 350 nm to about 700 nm). Examples of materials suitable for use asanti-reflective layers include, but are not limited to, certain metalfluorides, metal oxides, and metalloid oxides, such as magnesiumfluoride (MgF₂), lithium fluoride (LiF), calcium fluoride (CaF₂), sodiumfluoride (NaF), silicon dioxide (SiO₂), yttrium oxide (Y₂O₃), andhafnium oxide (HfO₂), as well as alternating combinations of two or moreof the foregoing.

In some embodiments, the anti-reflective layer 494 is a high-performanceanti-reflective layer. One non-limiting example of a high-performanceanti-reflective layer is a material having an average reflectance (Ray)of less than or equal to 0.3% (Ray≤0.3%) over a wavelength range of 380nm to 710 nm, and a maximum reflectance (Rmx) of less than or equal to1% (Rmx≤1%) over the wavelength range of 380 nm to 710 nm. Thehigh-performance anti-reflective layer may comprise one of (or acombination of two or more of) the fluoride materials and/or oxidematerials noted above. Certain materials that are otherwise effective asanti-reflective layers exhibit unacceptably high autofluorescence due topresenting color centers, which are due to the presence of rare earthelements and other impurities in the material. One example of such amaterial is MgF₂. In some embodiments, the high-performanceanti-reflective layer comprises one or more MgF₂ layers (or layers ofother AR materials exhibiting autofluorescence) separated in analternating fashion with layers of a different composition such as, forexample, SiO₂. The thickness of the MgF₂ layer (or other AR materialexhibiting autofluorescence), or the overall thickness of the MgF₂layers collectively, is minimized to reduce autofluorescence. Suchmaterials are referred to herein as low-autofluorescence anti-reflectivematerials. In some embodiments, the high-performancelow-autofluorescence anti-reflective layer may be a coating materialavailable from Penn Optical Coatings, LLC, Pennsburg, Pa., USA, havingthe product designations MD-FS-1001, MD-15-15771-01-A, andMD-15-15771-01-B.

Typically, the anti-reflective layer 494 is a thin layer (on the orderof micrometers) applied by a vacuum deposition process. In someembodiments, the anti-reflective layer 494 may be formed by applying theanti-reflective material to the substrate 490, followed by forming thepatterned mask 492, followed by again applying the anti-reflectivematerial so as to conformally coat the patterned mask 492. As a result,the patterned mask 492 is embedded in the anti-reflective coating 494,as illustrated in FIG. 4. In other embodiments, the patterned mask 492may first be formed directly on the substrate 490, followed by applyingthe anti-reflective coating 494 so as to conformally cover the patternedmask 492 and exposed surfaces of the substrate 490.

The same or similar type of anti-reflective layer may be applied to thesurfaces of any of the other optical components of the confocalmicroscope 100 (FIG. 1), particularly optical components positioned suchthat incident light reflected from them may propagate back to the lightdetector 112. For example, in some embodiments the window 132 and/or thefield lens 136 may have conformal anti-reflective layers. Moreover, thesubstrate of the window 132 and/or the field lens 136 may be alow-autofluorescence substrate as described above.

It will be understood that in the fabrication methods described herein,prior to forming any layer on an underlying surface, additional stepsmay be taken as needed to prepare the underlying surface such as, forexample, cleaning, etching, planarizing (e.g., lapping or polishing),dehydration, surface functionalization (e.g., adhesion promotion,passivation, etc.), etc. Such additional steps may or may not result inthe formation of an additional, identifiable thin film on the underlyingsurface. Such additional thin films, if present in practice, are notspecifically shown in the drawing figures.

As noted, the above-described approaches taken to reduce reflectance andautofluorescence from the spinning disk 416 and/or other opticalcomponents reduce the “ghost” light captured by the light detector 112(FIG. 1). This enables an increase the signal-to-background ratio for agiven exposure time, or a reduction in the exposure time required toattain a desired signal-to-background ratio. Reducing the backgroundimproves image contrast and improves assay results. Reducing theexposure time reduces the image acquisition time and thus increasessystem throughput.

FIG. 5 is a perspective view of an example of a lens doublet 500according to some embodiments. One or more of the optical components ofa microscope, such as described above and illustrated in FIG. 1, may beconfigured as a doublet, one non-limiting example being the field lens136. The doublet 500 includes a first substrate or lens 504 and a secondsubstrate or lens 508 bonded to the first substrate 504 at an interface512. Thus, the doublet 500 includes a first outer surface 516corresponding to the outer surface of the first substrate 504, and asecond outer surface 520 corresponding to the outer surface of thesecond substrate 508. The thickness of the doublet 500 through which theoptical axis passes is defined between the first substrate 504 and thesecond substrate 508. The first substrate 504 and the second substrate508 are bonded together at their inner surfaces, thereby defining theinterface 512. The first substrate 504 and the second substrate 508 mayhave different lens configurations. For example, in the illustratedembodiment the first substrate 504 is a crown lens and the secondsubstrate 508 is a flint lens, cooperatively forming an achromaticdoublet.

In some embodiments, the first substrate 504 and/or the second substrate508 is a low-autofluorescence substrate, and thus may be composed of alow-autofluorescence substrate material as described above. Therespective materials of the first substrate 504 and the second substrate508, whether or not those materials are low-autofluorescence materials,may have the same composition or different compositions. The TABLE belowprovides six examples of a field lens doublet in which one or both ofthe first substrate 504 and the second substrate 508 are composed of alow-autofluorescence material.

Surface Radius Thickness Glass 1 91.62609 3.6 N-KZFS8 2 −50.3292 2 N-BK73 62.66428 1 69.65163 3.6 N-KZFS8 2 −69.1317 2 N-BK7 3 47.8353 1109.2583 2 N-KZFS5 2 −64.786 3.6 Silica 3 83.03165 1 115.012 3.6 Silica2 −61.206 2 N-KZFS5 3 −154.59 1 75.51843 3.6 N-SF57HT 2 −923.263 2N-FK51A 3 62.11698 1 −110.061 2 N-FK51A 2 227.5433 3.6 N-SF57HT 3−56.2784

Generally, the first substrate 504 and the second substrate 508 may bebonded together by any suitable optically transparent bonding agent(e.g., adhesive, glue, cement, etc.). The bonding agent may beconsidered to be a film located at the interface 512. In someembodiments, the bonding agent is a low-autofluorescence bonding agent.Examples include, but are not limited to, silicone-based bonding agents.Other examples include, but are not limited to, mercapto-ester basedoptical adhesives and optical adhesives available from Norland ProductsInc., Cranbury, N.J., USA, having the product designations NOA 63, NOA81, and NOA 83.

In some embodiments, the doublet 500 includes an anti-reflective layerconformally coating the first outer surface 516 and the second outersurface 520. The anti-reflective layer may be a high-performanceanti-reflective layer or a low-autofluorescence anti-reflective layer asdescribed above.

For purposes of the present disclosure, it will be understood that whena layer (or film, region, substrate, component, device, or the like) isreferred to as being “on” or “over” another layer, that layer may bedirectly or actually on (or over) the other layer or, alternatively,intervening layers (e.g., buffer layers, transition layers, interlayers,sacrificial layers, etch-stop layers, masks, electrodes, interconnects,contacts, or the like) may also be present. A layer that is “directlyon” another layer means that no intervening layer is present, unlessotherwise indicated. It will also be understood that when a layer isreferred to as being “on” (or “over”) another layer, that layer maycover the entire surface of the other layer or only a portion of theother layer. It will be further understood that terms such as “formedon” or “disposed on” are not intended to introduce any limitationsrelating to particular methods of material transport, deposition,fabrication, surface treatment, or physical, chemical, or ionic bondingor interaction. The term “interposed” is interpreted in a similarmanner.

In general, terms such as “communicate” and “in . . . communicationwith” (for example, a first component “communicates with” or “is incommunication with” a second component) are used herein to indicate astructural, functional, mechanical, electrical, signal, optical,magnetic, electromagnetic, ionic or fluidic relationship between two ormore components or elements. As such, the fact that one component issaid to communicate with a second component is not intended to excludethe possibility that additional components may be present between,and/or operatively associated or engaged with, the first and secondcomponents.

It will be understood that various aspects or details of the inventionmay be changed without departing from the scope of the invention.Furthermore, the foregoing description is for the purpose ofillustration only, and not for the purpose of limitation—the inventionbeing defined by the claims.

What is claimed is:
 1. A microscope, comprising: a light sourceconfigured for producing excitation light; a sample stage configured forsupporting a sample at a sample plane; a light detector configured foracquiring an image of the sample; and optics defining an excitationlight path from the light source to the sample plane, and an emissionlight path from the sample plane to the light detector, wherein theoptics comprise at least one spinning disk comprising alow-autofluorescence substrate.
 2. The microscope of claim 1, whereinthe spinning disk is positioned at an intermediate image plane in theexcitation light path and the emission light path.
 3. The microscope ofclaim 1, wherein the low-autofluorescence substrate comprises ahigh-performance anti-reflective layer coating.
 4. The microscope ofclaim 3, wherein the anti-reflective layer coating is alow-autofluorescence anti-reflective layer.
 5. The microscope of claim1, wherein the low-autofluorescence substrate comprises a firstsubstrate and a second substrate bonded to the first substrate by abonding agent.
 6. The microscope of claim 5, wherein the secondsubstrate is a low-autofluorescence substrate.
 7. The microscope ofclaim 5, wherein the bonding agent is a low-autofluorescence bondingagent.
 8. The microscope of claim 1, wherein the spinning disk comprisesa plurality of apertures, and wherein the optics define an excitationlight path from the light source, through the apertures of the spinningdisk and to the sample plane, and an emission light path from the sampleplane, through the apertures and to the light detector.
 9. Themicroscope of claim 8, wherein the spinning disk comprises a patternedmask in which the apertures are formed.
 10. The microscope of claim 8,wherein the plurality of apertures are arranged along a plurality ofspiral paths.
 11. The microscope of claim 10, wherein the plurality ofapertures is selected from the group consisting of: the plurality ofapertures is a plurality of slits, each slit continuously extendingalong a corresponding one of the spiral paths; the plurality ofapertures is a plurality of groups of pinholes, wherein the pinholes ofeach group are arranged and spaced from each other along a correspondingone of the spiral paths; and the plurality of apertures is a pluralityof groups of slits, wherein the slits of each group are arranged andspaced from each other along a corresponding one of the spiral paths.12. The microscope of claim 9, comprising an anti-reflective layercoating the low-autofluorescence substrate and the patterned mask. 13.The microscope of claim 1, wherein the optics comprise a componentselected from the group consisting of: an emission filter in theemission light path; an emission filter in the emission light path, theemission filter configured for transmitting a selected fluorescentwavelength and substantially blocking other wavelengths; an emissionfilter in the emission light path, and comprising thelow-autofluorescence substrate; an emission filter in the emission lightpath, and comprising the low-autofluorescence substrate and ananti-reflective layer coating the low-autofluorescence substrate; awindow in the emission light path; a window in the emission light path,and comprising the low-autofluorescence substrate; a window in theemission light path, and comprising the low-autofluorescence substrateand an anti-reflective layer coating the low-autofluorescence substrate;a field lens in the emission light path; a field lens in the emissionlight path, and comprising the low-autofluorescence substrate; a fieldlens in the emission light path, and comprising the low-autofluorescencesubstrate and an anti-reflective layer coating the low-autofluorescencesubstrate; a field lens in the emission light path, and comprising afirst substrate and a second substrate bonded to the first substrate bya bonding agent, wherein at least one of the first substrate and thesecond substrate is the low-autofluorescence substrate; a field lens inthe emission light path, and comprising a first substrate and a secondsubstrate bonded to the first substrate by the low-autofluorescencebonding agent, wherein at least one of the first substrate and thesecond substrate is a low-autofluorescence substrate; a field lens inthe emission light path, and comprising a first substrate, a secondsubstrate bonded to the first substrate by a bonding agent, and ananti-reflective layer coating the first substrate and the secondsubstrate; and a combination of two or more of the foregoing.
 14. Amethod for acquiring microscopic images of a sample, the methodcomprising: operating the microscope of claim 1 to irradiate a samplewith excitation light and collect emission light emitted by the sample,wherein the excitation light and/or the emission light passes through anoptical component composed at least partially of a low-autofluorescencesubstrate, the optical component being a spinning disk.
 15. The methodof claim 14, wherein the emission light is fluorescent emission light.16. An optical component for a microscope, the optical componentcomprising at least a spinning disk composed at least partially of alow-autofluorescence substrate.
 17. The optical component of claim 16,comprising a high-performance anti-reflective layer coating thelow-autofluorescence substrate.